A widely studied supplement to cardioplegia solutions, glutamate plus aspartate, is thought to have beneficial effects because of its effects on the citric acid cycle. A recent report (J. Clin. Invest. 92: 831-839, 1993) found no effect of glutamate plus aspartate on metabolic pathways in the heart, but the experimental conditions did not model clinical cardioplegia. The purpose of this study was to determine the effects of glutamate and aspartate on metabolic pathways feeding the citric acid cycle during cardioplegic arrest in the presence of physiological substrates. Isolated rat hearts were supplied with fatty acids, lactate, pyruvate, glucose and acetoacetate in physiological concentrations. These substrates were enriched with 13C which allowed a complete analysis of substrate oxidation by 13C NMR in one experiment. Three groups of hearts were studied: arrest with potassium cardioplegia, arrest with cardioplegia supplemented with 13 mM glutamate and 13 mM aspartate, and a control group without cardioplegic arrest. We found that in potassium-arrested hearts, the contributions of fatty acid and lactate to acetyl-CoA were reduced, and acetoacetate was the preferred substrate for oxidation in the citric acid cycle. The addition of aspartate and glutamate in the presence of cardioplegic arrest did not further alter substrate utilization patterns substantially, although acetoacetate utilization was somewhat lower compared to simple cardioplegic arrest. When 13 mM [U-13C] glutamate or 13 mM [U-13C] aspartate were supplied as the only 13C-labeled compounds, little enrichment in citric acid cycle intermediates could be detected. We conclude that glutamate and aspartate when added to potassium cardioplegia have relatively minor effects on citric acid cycle metabolism.